RNA replication-independent, DNA linearization-dependent expression of reporter genes from a SARS-CoV-2 replicon-encoding DNA in human cells

Friedhoff, R., Elfayres, G., Mérindol, N., Desgagné-Penix, I. et Berthoux, L. (2024). RNA replication-independent, DNA linearization-dependent expression of reporter genes from a SARS-CoV-2 replicon-encoding DNA in human cells. PLoS ONE, 19 (8). Article e0300491. ISSN 1932-6203 DOI 10.1371/journal.pone.0300491

Prévisualisation

PDF Disponible sous licence Creative Commons Attribution. Télécharger (2MB) | Prévisualisation

Résumé

Abstract

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

Type de document:	Article
Mots-clés libres:	Adenosine Monophosphate Alanine Chromosomes Artificial Bacterial COVID-19 DNA-Directed RNA Polymerases Genes Reporter Green Fluorescent Proteins HEK293 Cells Humans Promoter Regions Genetic Replicon RNA Replication RNA, Viral SARS-CoV-2 Transfection Viral proteins Virus replication Green fluorescent protein Luciferase Remdesivir Restriction endonuclease RNA polymerase Adenosine phosphate Bacteriophage T7 RNA polymerase DNA directed RNA polymerase Viral protein Virus RNA Article Bacterial artificial chromosome Centrifugation DNA transfection Electroporation Escherichia coli Flow cytometry Fluorescence microscopy Gene expression Genetic transfection HEK293T cell line Human Human cell Luciferase assay Lung adenocarcinoma Polymerase chain reaction Protein expression Reverse transcription polymerase chain reaction SARS coronavirus Western blotting Coronavirus disease 2019 Genetics HEK293S cell line Metabolism Physiology Promoter region Reporter gene Severe acute respiratory syndrome coronavirus 2 Virology
Date de dépôt:	22 avr. 2025 19:20
Dernière modification:	22 avr. 2025 19:20
Version du document déposé:	Version officielle de l'éditeur
URI:	https://depot-e.uqtr.ca/id/eprint/11864

Actions (administrateurs uniquement)

Éditer la notice